Group Title: effect of high temperature on the free amino acids of common pea (Pisum sativum L.) /
Title: The effect of high temperature on the free amino acids of common pea (Pisum sativum L.)
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Title: The effect of high temperature on the free amino acids of common pea (Pisum sativum L.)
Physical Description: vii, 122 leaves : ill. ; 28 cm.
Language: English
Creator: Shokraii, Esmail Hosseini, 1935-
Publication Date: 1965
Copyright Date: 1965
 Subjects
Subject: Plants, Effect of temperature on   ( lcsh )
Growth (Plants)   ( lcsh )
Plant physiology   ( lcsh )
Peas   ( lcsh )
Botany thesis Ph. D
Dissertations, Academic -- Botany -- UF
Genre: bibliography   ( marcgt )
non-fiction   ( marcgt )
 Notes
Thesis: Thesis (Ph. D.)--University of Florida, 1965.
Bibliography: Includes bibliographical references (leaves 115-121).
Additional Physical Form: Also available on World Wide Web
General Note: Typescript.
General Note: Vita.
Statement of Responsibility: by Esmail Hosseini Shokraii.
 Record Information
Bibliographic ID: UF00097912
Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
Resource Identifier: alephbibnum - 000560612
oclc - 37463431
notis - ACY6171

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THE EFFECT OF HIGH TEMPERATURE

ON THE FREE AMINO ACIDS OF COMMON PEA
(PISUM SATIVUM L.)
















By
ESMAIL HOSSEINI SHOKRAII


A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY


UNIVERSITY OF FLORIDA
April, 1965


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S ACKNOWLEDGMEW1S -


The author wishes to express his best gratitude and

thanks to Dr. David S, Anthony, Chairman of the Committee,

for his kindness and continuous supervision during the

pursuit of this degree, especially during the course of
Research and preparation of this dissertation.
The assistance of the members of the committee,

Dr. G. Ray Noggle, Dr. R. H. Biggs, Dr. A. D. Conger and
Dr. G. J. Fritz is also gratefully acknowledged.

Appreciation is also extended to the Fulbright
Commission, the American Friends of the Middle East,
University of Florida sand the Department of Botany for
. their very generous fellowships and assistantships which .
made it possible for the author to extend his graduate
studies at the University of Florida.











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TABLE OF CONTENTS

Page
ACKNOWLEDGMENTS .............. ii

LIST OF TABLES. . .. . . . . . v

LIST OF FIGURES. .... . . . . vi

INTRODUCTION. . . . . . . . . . . 1

LITERATURE REVIEW . . . . . . . 9

A. Effects of high temperature on
microorganisms. . . . . . 9
1. Higher plants . . . . . . 11

MATERIALS AND METHODS . . . . . . . 22

A. Plant material and growing conditions 22
B. Sampling. . . . . . . . 23
C. Preparation of extracts for amino
acid analysis. . . . . . 24
D. Purification of extracts by ion-
exchange resin . ..... 24
B. Qualitative identification of amino
S acids in the extracts. . . . 25
F. Quantitative determination of soluble
amino acids in pea extracts. . 26
G. Protein determination . . . . 37
H. Chemical treatment techniques .. . .40

SAMPLING AND PHYSIOLOGICAL AGE. . . . . . 43

RESULTS AND DISCUSSION' .. .. . . 47

I. Growth Characteristics of Plants Under
Optimum and High Temperature Conditions. 47
II. Amino Acid Analyses of Peas Under Optimum
and High Temperature Conditions. . 65

III. Protein Estimation. . . 88
i .. . 88


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IV. "'he Effects of Adeod Meta

TemperatiuZe : . .


SUMMARY AND CONCLUSIONS.


LITERATURE CITED .


BIOGRAPHICAL- S OTCH. .

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LIST OF TABLES

Table Page
1. Growing Conditions in Growth Chamber . . 23

2. Preparation of Buffers .. . ... .. 30

3. Percentage Recovery of Amino Acids from the
Long andShort Column. . . .. . . 33
4. Composition of Metabolite Solutions Sprayed on
Leaves . . . . . . . 42

5. Amino Acid Content of Leaves First Period of
Sampling . . . . . . . . . 70

6. Amino Acid Content of Leaves Second Period
of Sampling . . . . . . . 73

7. Amino Acid Content of Root Second Period of
Sampling . . ... . . . . . 76

8. Amino Acid Content of Leaves Third Period of
Sampling . . . . . . . . . 81

9. Amino Acid Contet of 6Leaves Fourth Priod

10. Amino Acid Cotntn' of Leaves Fifth Period of
Sampling * *** 89

11. Protein Content of Leaves., ., .. ., . 102






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LIST OF FIGURES

Figure Page

1. Daily height increase of pea plants under two
temperature regimes . . . 49
2. Growth (height increase) of the pea plants
under high and optimum temperature conditions
as compared with the groups of plants which
were depistillated (in order to stop fruit
formation) at the optimum temperature condition 51

3. Daily height increase at optimum condition
(pistil removed). . . .. 54

4. Total fresh weight increase and the weekly
increase of fresh weight at high and optimum
temperature conditions. . . . . 58

5. Total dry weight increase and the weekly
increase of dry weight at high and optimum
temperature conditions.. . .. . . . 61
6. Increase in percent dry weight at different
stages of growth at optimum and high
temperature conditions. . . . 64

7. Shoot-root weight ratio versus time .. 64
8. Chromatograms comparing amino acid constituents
of leaves of pea plants under optimum and high
temperature conditions. . . .75

9. Chromatograms of amino acid constituents of
roots at optimum and high temperature
conditions. * 78
10. Chromatograms of amino acid constituents of
leaves at optimum and high temperature . . 83
11. Chromatograms o6f mino acid constituents of
leaves attopti ~u and high temperature. .. 87

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12. Chromatograms of amino acid constituents
leaves at optimum and high temperature.
(Fifth period of sampling) .. . ..


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13. Histograms comparing the changes in the amount
of aspartic acid, asparagine, glutamine and
glutamic acid in the leaves of plants grown
under optimum and high temperature during five
successive weeks 6f growth . . .

14. Histograms comparing the changes in the amount
of alanine,' erine, glycine and threonine in
the leaves of plants grown under optimum and
high temperature during five successive weeks
of growth. ... < -. 4 . . . . . .

15. Histograms comparing the changes in the amount
of homoserine, valine, leucine and isoleucine
in the leaves of plants grown under optimum
and high temperature during five successive
weeks of growth. . . . . . .

16. Histograms comparing the changes in the amount
of -aminobutyric acid, lysine, histidine and
phenylalanine plus tyrosine in the leaves of
plants grown under optimum and high
temperature during five successive weeks of
growth . . . . . . . . . .

17. Total soluble protein content of leaves of the
plants grown under optimum and high tempera-
ture during five successive weeks of growth..

18. Total amino acid content of leaves of plants
grown under optimum and high temperature
conditions during five successive weeks of
growth *..* *.** *,* ,


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IN PRODUCTION


in spie of the large amount of descriptive and
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morphological in~foation *aabl e do od ing the effects
-of high temperature on plant growth and development,

especially with the invention of modern climatic control
facilities such as growth chambers and the phytotron (78),
little is known of the biochemical effects of high tempera-
ture on plants. Further research in this area should be
quite interesting and hopefully fruitful from agricultural
and physiological points of view. There are many economic
plants that are restricted to certain areas mainly because
of their requirement for a certain temperature range.
Further studies in this field seem to be quite
necessary to develop the fundamental information needed to
establish rational approaches to such problems as treatment
of climatic lesions and the adaptation of different plants
of temperature zones to tropical and sub-tropical areas or
vice versa. Such research wold provide basic information
about one of the most important variables of growth in
plants a variable which has not been thoroughly invest.i-

.. ated (28,29).
her is little information concerning t.he major




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factors that limit plant growth and development at high
temperatures, but in general onoe an cite the hypothesis
that plant growth or stage of development may be altered
.through limitation of the velocity of a single reaction.
Thus, limitation of a single or a few necessary critical
substances may cause the changes in growth and development,
According to the present concepts of molecular
biology, the destruction of proteins, DNA or RINA might cause
limitation of growth at high temperature. (High temperature
might more correctly be called supra-optimal temperature,
but is seldom so described in the literature or in the
present paper.) Thermal studies qn soluble ENA in vitro
revealed the maximum temperature limit for.acceptance of
amino acids is around 750 0. he '"melting apart" of the
two strands of DNA at an elevated temperature is also widely
reported, and it is believed that the temperature at which
strand separation of D~PA cold occur is in te range of 70 C.
to 900 C. (i8).
The greatest range of atadatitin ; temperature is
found in microorganisms, where some species are found that ,
are able to grow at .40 C* and others to +70 0. (30).
Several fungi may also grow below 00 0., but their upper
limit of growth is 400 C. to 500 0- %Of course, duration of
temperature is important since, for a short period of time,
some spores can tolerate temperatures as high as 1350 C.



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(The temperature limits of growth and development in
poikilothermic organism are tabulated in the "Handbook of
Biological Data" by Spector (68).)
The highest temperature at which organisms grow on
the earth's surface are found. in hot spring areas in the
Yellowstone region of the United States, in Japan, New
Zealand and Iceland (10). There are numerous reports of
algae and bacteria found in these places where the highest
temperature is 730 0. Studies by Koffler (33) on life in
hot springs indicate that the thermophilic organisms have
evolved some kind. of protein that is much more thermostable
than that of .mesophi3ic species.
Flowering plants appear to have temperature limits.
similar to those of Tungi As they, pan grow at several
degrees below zero and at temperatures above 500 C. The
temperature limits of desertic plants have been discussed
briefly by Kurtz (35) and the maximum is found in desert
areas .of-the United States. Among the members of the cactus
family there are species like Opuntia that grow at tempera-
tures as high as 580 0G In Australian deserts, some species
of Atriplex, e.g. A. vesicarium, exhibit a maximum rate of
photosynthesis at 40 0. to 50 Most of these desertic
plants are obligate thermophiles. The lethal high tempera-
ture according to Lorenz (48) varies with: 1) species of
plant; 2) duration and method of.applioation of temperature,

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since gradual increase of temperature has a much less harmful

effect; 3) age of tissue and organ; 4) time of exposure to

high temperature (day or night); 5) part of plant (root or

shoot), tops of plants are more resistant than roots. This

last mentioned phenomenon may be due to the presence of more

protective tissue around the stem and leaves rather than to

difference in heat tolerance of protoplasm.

The damage caused by higher than optimum temperature

on flowering plants and lower plants. seems to be mediated in

large part by an effect on some chemical reactions-within

the plants. However, only in a very few cases has the

biochemistry of the plants been studied in relation to high

temperature as was mentioned before (36442).

Although there is little inforzation'on reactions
affected, there are observations of biochemical differences

which have been attributed to high' temperature such as:

change of amount of aromatic compounds in flowers and fruits

(especially in the tea plant), decrease of soluble sugar,

formation of pigments, .and changes in pigment concentration

of leaves, flowers and fruits. The red and pink colors
often become more pronounced at lower temperatures since

anthocyanin pigment formation actually is responsive to
sugar content which in turn is. affected by temperature.

Growth, as determined by fresh weight and dry weight,

reduces at high temperatures. The cause of this reduction



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is not really understood, although several explanations are,
offered. Many investigators believe that decrease in growth
of plants at .temperatures above optimum is partly due to
limitation of translocation. Temperature affects trans-
location (77), Maximum translocation occurs at about 100 0.
in many.plants. Thus, indirectly, root weight might change
with high temperatures as root weight is quite responsive to
rate of translocation. High temperature causes an increase
in respiration greater than an increase in photosynthesis,
and this would cause a decrease of growth mostly due to
reduction in sugar and other reserve materials (4,20,26,47).
Nightingale (59) reports that orchard trees are able to
accumulate more carbohydrate at moderate rather than at high
temperatures.\ In'sugar beets there is an inverse correlation
between the amount of sugar and the temperature of the
environment. !Finally, among the other effects of high
temperature that.could affect, growth are mentioned suscepti-
bility of plants to disease and also the increased phyto-
toxicity of toxic compounds, e.g. 2,4 D is more toxic at
high temperature than at low temperature.
An interesting and complex interaction between plants
and temperature s diirnal and annual thermoperiodicity
which are very common among ,he higher plants. In general,
the optimum temperature for night is lower than for day.
Spring annuals die when subjeoe, t.6 a high night temperature







6

of about 25 0., although they will grow well at a day

temperature of 25 C.

It has been observed (34,78,79). that in certain
plants resistance to cold can be increased by growing them

under a condition of alternating light and dark in which the

daily rhythm is not a 24 hour cycle, but somewhat longer.

On the other hand, the same plant can be made resistant to
high temperature damage by growing it under a daily cycle

of less than 24 hours.

All these facts suggest that most plants do have a
kind of timing mechanism or a diurnal rhythm which seems to

coincide with a 24 hour period (8) at optimum temperature.

At low temperature the cycle of diurnal rhythm is slower,

and there is a failure of synchrony between the plant and

outside environment. This in turn can cause metabolic im-

balance and possible visible disturbances.

In general, the factors that have been suggested as

responsible for the effects of higher than optimum tempera-

ture on plants could be summarized as follows (42):

Change (decrease) in availability of gases.-In some
microorganisms it has been shown that high temperature has

a direct effect only on the availability and amount of

gases dissolved in theq culture medium. .

Acceleration of breakdown of enzymes, vitamins and
other metabolites.-Again, extensive experiments with



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microorganisms in controlled culture media have shown that
the requirement of the organism for a growth factor or enzyme.
will rapidly increase with elevation of temperature.
Changes in the balance of interconnected reactions.-
Temperature extremes thus might cause accumulation of some.
toxic products, or reduction of necessary factors. In
either case, the regulation of metabolism would change.
Consequently, growth would be affected.
Enzyme inactivation.-Studies with microorganisms
support the idea that simple inactivation of a single
thermolabile enzyme could result in a lesion due to higher
than optimum temperature, e.g.., the high heat sensitivity
of nicotinamide adenine dinucleotide (NADH2) oxidase has
been demonstrated in microorganisms, A similar effect of
high temperature on enzymes of higher plants could exist.
Inhibition of enzyme formation.-The effect of high
temperature inhibition of the formation of an enzyme can be
due to either inactivation and destruction of RNA, or the
inhibition .of activityy of the operator gene for enzyme
formation.
Enzyme activation.-Several experiments show that heat
activation of an enzyme might occur, since heat can destroy
a thermolabile iOlibtor of an enze aAg i t us cause indi-
rectly an activatio f anf enzyme reaction.

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8

In biochemical studies of higher than optimum tempera-

ture it is important to select a plant that has the following

qualifications:.

It should be sensitive to high temperature.

It must have a rather short life span to make large

numbers of replicates possible in a reasonable time.

It should be rather small in size to be carried and

handled easily in controlled conditions.

It.should be able to grow well under controlled

conditions and also in artificial media.

There are other important points to consider in addition to

choice of plant material. The method of application of

high temperature must be considered. Is high temperature

going to be given only at the beginning of germination or

during maturity?. Another question to consider is the

duration and intensity of the high temperature stress.










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LITERATURE REVIEW

A. Effects of high temperature on microorganisms

The largest body of work on the biochemical-effects

of temperature considered to be higher than optimum has been

done using microorganisms.

It has been shown in a number of microorganisms that
the reduction in growth rate which occurs at higher than

optimum temperatures could be prevented or maximum growth

could be restored by supplying a specific chemical (meta-
bolite) in the growth medium. If the temperature was much

Higher than optimum, addition of that specific metabolite

had to be greatly increased or a combination of two or

three different metabolites was required to restore the

growth of the microorganism to or toward normal (42).

If the temperature was dropped back from supra-
optimum to optimum, maximum growth was restored without the

further addition of any of the metabolites.

The so-called temperature sensitive mutants of
Neurospora crassa and Escherichia coli have been extensively
studied (2,51,52,57). It has been shown that Neurospora

crassa ca.o.grow quite well at temperatures up to 55* C. to
40 C., however, there is a mutant of Neurospora which grows

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well at or below 250 C. Its growth rate decreases markedly
with further increase of teiperate i~ at 28 0. growth
stops completely. Growth can he restored at 28 0. in this

microorganism by addition of 2.5 x 10 gm of riboflavin to
each liter of culture solution. There seems to be a perfect
riboflavin-temperature-growth iesp6nse interaction (57).
Other mutants have been found that can be protected
against the development of high temperature lesions by
simultaneous supplementation with adenine, pyrimidine and
methionine in their media (55). A temperature sensitive
uracil-requiring mutant has also been isolated (25) where
there is a complete block for uracil synthesis at high
temperature. Addition of uracil to the growth medium of
this organism causes normal growth even at temperatures

higher than optimum. Temperature-dependent tyrosine re-
quirement in Neurospora also has been reported (24) and was
interpreted as a result of lower thermostability of the
mutant enzyme. Addition of typrosine prevented the defect.
In E. coli, panthothenate-requiring temperature-
sensitive mutants have been isolated. In this microorganism,
the enzyme responsible for the condensation of 8-alanine and
pantoic acid to form pantothenic acid is apparently more heat
labile than other enzymes. Thus, the addition of pantho-
thenic acid to cultures of this temperature-sensitive mutant
allows them to grow at elevated temperatures in a manner









identical with normal strains. Amongth 6rganic compounds
which have been found to reduce high temperature damage in
one or more of a large group of microorganisms and fungi by
several investigators (3 1616)one may mention:

methionine, glutamic acid, thiamin, biotin, a number of
other amino acids, and other members of the vitamin B complex.


because of high temperature, but, on the other hand, Hertman
(19) has seen the induction of impairment of some bio-

chemical processes. For instance, in Pasteurella pestis at
7s C. (optimum temperature is 27( C.) the formation of
pestioin stops. At high temperature, addition of amino
acids such as leucine and/or isoleucine relieves the high

temperature effect and pesticin can be produced the same

as under optimum temperature.

B. Higher plants
Some of the earliest work on the biochemical effects
of abnormal temperature was by James Bonner (7) who proposed
the term "climatic lesion" for the first time. His concept
was that at higher or lower than optimum temperature one or

several biochemical events cannot take place simply because
the enzymes responsible for those biochemical syntheses are
more thermolabile than other enzymes. Therefore, after a
short time there would be a shortage of those particular
metabolites. Bonner proposed that the shortage of such

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metabolites is the cause of the "climatic lesion" and that
the lesion might be cured by external supplementation of the
particular compound or compounds which were absent. In other
words, under unfavorable temperature conditions, green plants

become heterotrophic for one or more specific metabolites.
Bonner tested his theories by experiments with Cosmos (6).
However, his published data are all concerned with the
effects of low temperature. He found the addition of thiamin
in the nutrition medium of the Cosmos caused an increase of
growth as measured by fresh weight at temperatures lower

than optimum. His results were most'significant where dry
weight was as low as 5 per cent of that' under optimum
conditions. Under these circumstances, thiamin caused a
40 per cent increase in dry weight. At a temperature where
dry weight was half of maximum, the increase due to thiamin
addition was 20 per dent. Addition of thiamin at the optimum
temperature had no stimulatory effect on growth. The con-
clusion was made that at low temperature, inhibition of
growth of the Cosmos plant can be overcome by an external
source of thiamin. This is the first recorded successful
chemical prevention of a climatic lesion. Later work on
prevention of low temperature damage on eggplant (solanum
melongens) (32) has shown a low temperature (14 C. night;

200 0. day) inhibition of growth can be overcome with a
mixture of ribosides sprayed on the leaves. While at optimum



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S 'tempera- ture , a) r sides ha little ,



or no effect. it has been shown tha
temperartua~e (0 CiF~~tieht temperature rbosles iaeo little
se*ton and aso- in exlsd eto e eas ,the addition
or no effect h growth mediu decr ease ben hio thate -
soaking of seeds in a solu ."on of nacQtin c acid caused
significant increase in tolerance of seedlings to low
temperature inhibition of growth (32)Y
The alleviation of a high temperature lesion appears
to have been accomplished for the first time by Galston and
Hand (15). They showed that, in etoilated pea epicotyl
sections and also in excised etoilated leaves, the addition
of adenine to the growth medium decreased the high tempera-
ture inhibition of growth. Adenine stimulation of growth
at optimum temperature was only half as much at high tempera-
ture (35 C.). They concluded that inhibition of growth at
high temperature in the pea tissue was partly due to adenine
destruction or inhibition of its synthesis. Further work
by Galston (17) on growth of intact pea plants (var. Alaska)
in the Earhart Phytotron Laboratory did not show a complete
reversal, of high temperature-induced decrease of growth by
adenine supplementation where plants were kept at high night
temperature and optimum day temperature. Possibly the
failure of successful cure of high temperature lesions with
adenine in this case was due to the use of high night tempera-
ture as the experimental condition, whereas later it was
shown that the pe is more sensitive to high day temperatures
(16,). Later work of Lockhart (44) with the same variety of

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pea indicated that adenine had no significant effect on stem
elongation and growth or on onset of maturity of the whole

pea plant kept at 300 C. However, it should be noted that
the plants in this study were kept at a constant high
temperature (day and night).
Despite the inconsistencies with respect to adenine
supplementation cited above, it is possible that adenine may
have some importance; because the adenine level in high
temperature-resistant pea plants increased with high tempera-
ture conditions while in a temperature-sensitive strain,
adenine decreased (22).
Additional evidence that adenine deficiency may be a
cause of high temperature injury in plants was obtained by
McCune (54) with the common duckweed (Lemna minor). He
showed that this plant could be protected against high
temperature damage by the addition of adenine in the form
of adenosine to its culture medium. Guanosine also was
found to be effective.
Additional work, showing climatic lesions and their
chemical treatment in peas, was reported by Ketellapper and
Bonner (31). They found that pictures of essential metabo-
lites such as a vitamin B mixture, or a riboside mixture,
partially or completely prevented the reduction of growth
at high temperatures. Sucrose and vitamin C also were found
to be effective. Ketellapper (32) found that inhibition of




,+ - +' ,. + I + --

4 15

growth at a temperature a few degrees higher than optimum
could be completely stopped by a spray of sucrose on leaves
while for conditions several degrees higher than optimum
there was no significant .effect for sucrose. This indicated
that sucrose and other metabolite effects are temperature
specific. It seems probable that at least part of the high
temperature response is mediated through the chemical
machinery of the plant, although the experimental evidence
is often confusing.
The study and interpretation of climatic lesions due
to high temperature in plants is complicated by several
factors. For example, within a given genus and species
there may be strains or varieties showing both quantitative
and qualitative differences in response to temperatures.
Langridge (39,40), working with a wide range of strains of
Arabidopsis thaliana, showed that the inhibition of growth
at elevated temperatures varied markedly from strain to
strain. Further, he showed that the high temperature
inhibition of growth was prevented in certain Arabidopsis
strains by the addition of biotin, in another strain by
cytidine, in another by chol'ihe, while in still other strains
he was unable to prevent te'Uhigh temperature effect by
chemical meins, Ketellapper (32) also warned that the
effective substances for preventihghigh.temperature effects
may vary from species to species.and even from variety to
variety within a species.*
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Interpretation of negative results in experiments
involving attempted cure or reversal of climatic lesions

by chemical means is difficult since one may not be sure

whether the added compound actually reached the affected

tissue (32).

Another surprising complication even within a given

genus, species and variety in the study of high temperature

effects is the variation due to seed source. In research
with peas (var. Unica), Highkin (21,22) found that the

conditions under which the seeds had been grown altered the

effects of'subsequent temperature treatment. He found that

constant day and night temperatures maintained over several

generations successively reduced the vigor of progeny in a
cumulative sense. Further, seeds from plants that had been

maintained for a few generations at constant temperature did

not produce plants of normal vigor when planted at optimal

fluctuating temperatures until the second or third generation

under the optimum conditions. Subsequently, Ketellapper (32),

with the same pea variety, showed that the effect of high

temperature on growth is counteracted either by sucrose or
by a vitamin B mixture and a riboside mixture, depending on
seed source (i.e. temperature conditions under which seed

had been grown). These results suggested that the phenotype

is a combination of its genetic heritage, its present environ-

ment, and the environmental conditions under which its

parent grew (23,41).

ii". \ -






SI: 17


Langridge (42) believes a numberr of' these growth
stimulations by chemicals reported above for higher plants
is of poor reprodudibility 'andL 6f doubtful statistical
significance, thus is in contrast to ,the high temperature
lesions that have been observed among microorganisms. In
the work with Arabidopsis thaliana already described,
Langridge and Griffing (40) did obtain clearcut, significant
responses to chemical additions at high temperatures in
aseptic culture.
Another interesting aspect of high temperature effects
on plants from the biochemical point of view is the effect
of high temperature on the response to auxin and gibberellin.
Galston (16) and Lockhart (46) studied responses of plants
to hormones under high temperature conditions. A silkless
mutant variety of corn at 20 C0. day and 14i 0. night
temperature showed a 50 per cent elongation response to
applied indole acetic acid (IAA), whereas a normal variety
gave no response at this temperature regime. However, at a
higher temperature, 260 0. day and 200 0. night, the silkless
variety showed no response to IAA while the normal variety
gave a 16 per cent elongation response. Lockhart observed
that applied gibberellin on leaves of Alaska peas grown at
high temperature had a very significant effect on delaying
the high temperature acceleration of senescence which
usually accompanies the onset of maturity. Later, Lang

(37,38) showed that failure of Hyocyamus in flower formation
*: 1'*. ,: i :^ : :







18

at high temperature is apparently because of gibberellin

deficiency rather than anything else since Hyocyamus needs

a large amount of gibberellin to elongate the flower stalk.

It was shown that gibberellin sprayed on leaves of Hyocyamus

and some other plants can replace the need of low night

temperature which otherwise was necessary for flower for-
mation. Among the other works that can be considered
related to this problem the following papers need to be
mentioned. Yarwood (80,81) has studied acquired tolerance

of leaves to heat (acquired heat tolerance of microorganism'
has been known for some time (62)) and also has observed

translocation of heat injury from one leaf to another.

When leaves of a number of a species of plants were pre-

treated at a temperature of 500 C., they subsequently (12,
14 hours later) tolerated a temperature of 550 C. up to

three times as long as did the control leaves which were not

previously heated. Also, a temperature of 650 0. applied
unilaterally to plants killedithe healed leaves, but at the
same time the leaves in the other unheated side or branches

became injured withiat any possible direct.aieat transfer (81).
The above mentioned reports constitute rSeview of the avail-
able literature dealing with the problem of metabolic or

biochemical effects of high temperature on plants. From the
scattered but divergent results already in the literature,

it seems likely that there is not a s~ gle general chemical

*~ ~~ :.f *
*~i **,





* " ^ '.^' " ,' : .' 1'' -" ' '
/ 19

explanation for heat: injury, but rather there may be many
explanations in many plants. Obviously, much more informa-
tion is needed before any general or specific theory of
heat injury, backed by sblid evidence, can be elaborated.
One omission' in studies of high temperature injury
in plants became apparent from the literature review. To
our knowledge, with the ,partial exception of one paper in
the Russian literature (60), there is no published evidence
that anyone has attempted to mak, quantitative biochemical
analyses comparing the composition of plants grown at high
temperature with that of the same plant grown at optimum
temperatures. That is, there is no direct quantitative
measure of the biochemical effects of high temperature on
higher plants.
Thus, since technological advancement has recently '-, (-/
been made in isolating and identifying most of tho bio-
chemical constituents of plants, it seemed reasonabletthat l,' :
an organized investigation of the quantitative effects of ,
heat on the biochemistry of plants might advance our uAder-
standing more rapidly than spraying or adding to the plant
in "shotgun" fashion a variety of metabolites with the hope
that this sprayed mixture contained the substance which was
deficient under the high temperature condition. In such
experiments, there must be the further hope that the critical
-substance (or substances) can be taken up and translocated
to the site f need.

.. .. "t.' ' "' '







20


However, it was very difficult to decide which groups
of compounds should be studied in preference to other groups.
The final decision was to study amino acids and proteins.
This decision was made for the following reasons.
Davern (11) extended the work of Ketellapper in a
study of the effects of amino acids on the growth and
development of clover (Trifolium subterraneum), a cold
temperature requiring plant. It has been shown that spray
of casein hydrohysate on the plant under high temperature
has quite a significant effect on the growth, while the
effect on plants under optimum conditions is not significant.
Davern did not study the effect of each single amino acid
but he used three groups of amino acids; protein amino acids,
non-protein amino acid and ring-structure amino acids. The
first and second groups were found to be much more effective
(especially the first group).
Petinov (60), in a research with leaf tissue culture,
has shown that high temperature increased the amount of free
amino acids. He was not looking for a lesion, and his
analytical technique was only roughly quantitative (one-
dimensional paper chromatography). :
Earlier work of F. C. Steward (70) has shown the
effects of various nutrition conditions as well as light
and dark period and high night temperature on the free amino
acids of Mentha piperita mint plant). He has shown that




A Re has shown. hat




i 7. ... A-, f !

21

amino acids change drastically with changes in the environ-
mental and nutritional variables.

Research on a large group of microorganisms and lower
plants have shown that high temperature lesion and inhibition

of growth can be prevented or reduced by a number of amino
acids (5,27,66).
Levitt (44,45) suggested a possible correlation be-

tween frost hardiness and high temperature hardiness. He
postulated that both are concerned with protein structure.

In view of the available evidence, it seemed
reasonable, as a first quantitative approach, to examine

the free amino acids and proteins of plants at optimum and

supra-optimum temperature, hereafter usually referred to as
high temperature conditions.

















r



i .











MATERIALS AND METHODS

A. Plant material and growing conditions

Pea seeds (Pisum sativum variety Greater Progress and
Wanda)* were hand-graded for uniformity, soaked five hours
in tap water at room temperature and then sown in plastic
flats (15" x 12" x 6.5"). The planting medium was a mixture
of equal volumes of washed sand and vermiculite. The flats
were watered (2,000 ml) and were transferred to growth
chambers (Percival Growth Chamber Model E-57). Every day
prior to germination of seeds each flat received 500 ml of
distilled water. After the seeds germinated, extra seedlings
and abnormally small or large seedlings were discarded in
order to have a uniform group of seedlings. All flats were
thinned to 12 seedlings with approximately equal spacing.
Ten days after planting, plants were watered with 500 ml of
Hoagland's solution per flat every other day. The plants
under high temperature conditions were given additional dis-
tilled water occasionally as needed to eliminate water
stress. The temperature of nutrient solution was not ad-
justed beforehand to the temperature of growing conditions
(usually it was less, especially at the high temperature
condition). The light source was a combination of fluorescent

*Purchased from Joseph Harris Co., Inc., Moreton Farm,
Rochester 11, New or, Yr. ,,
S1 221 K.. 2.i -
, . .. .Irl i', ;,I : :,-.- ,'; - .







23

TABLE 1

Growing Conditions in Growth Chamber

Temperature Phototemperature Nyctotemperature
condition (16 hr.) (8 hr.) Light intensity

Optimum 1 3 + 1 17 i+1 1200 + 200 f.c.

High temperature 30 + 1 23 + 1 1200 + 200 f.c.
,, *




(cool white) and incandescent bulbs (Sylvania lamps). For

each experimental condition tested 60 plants were used. Ten

plants were selected at random after the first week of

growth for measurement of daily height increase.

B. Sampling,

Six plants were selected randomly for each extract.

First, they were washed free of rooting medium and weighed

immediately. The number of .leaves, height of shoot, total

and shoot fresh weight, root weight, shoot-root ratio, dry

weight and later number of buds, flowers and fruits were

measured. Alcoholic extracts were prepared from leaves,

roots, and stem. The plant tissues were cut into small

(0.5 cm) pieces and dropped into about 150 ml of 80 per cent
ethyl alcohol at room temperature. The alcohol was allowed

to remain in contact with the plant tissue during storage at

00 .. prior to analysis. (This storage period was from one

to three months.)I he time of original sampling-was the


.. . ..




1


24

same (8:.00 P.M.) in the two temperature conditions and

usually the time of sampling was 12 hours after watering.

Samples were taken once a week.

C. Preparation of extracts for amino acid analysis

The stored alcoholic extracts and the tissue residue

were homogenized thoroughly in a glass homogenizer and

vacuum filtered through Whatman #1 filter paper. The fil-

trate was concentrated in a Rinco flash evaporator under

vacuum at room temperature. The concentrated (now aqueous)

extract was centrifuged 15 minutes at maximum speed in a

clinical centrifuge (approximately lOOOg). The precipitate

was discarded. An equal volume of chloroform was added to

the supernatant with vigorous shaking to remove fats and

pigments. The chloroform layer was discarded. The defatted

aqueous extract prepared in this way was further concentrated

to 1 ml/1 gm wet weight of original plant sample.

D. Purification of extracts by ion-exchange resin

An ion-exchange column approximately 15 cm in length,

containing 10 gms Dowex 50, was washed several times with

distilled water, the Dowex 50 was regenerated by passing

80 ml 3 N HO1 through the column. Since the volume of the

15 cm resin column was 13 nl, the 80 ml HC1 rinse was about

six column volumes (67). The HC1 was washed out by passing

200 or more ml of water through 'the column until the column

was chloride free (checked with AgTNQ). The plant extracts
,1 ! '. \ : : * :.' > '


i




C,


25

were passed through the' washed column in ice cold condition
(column was placed in ice and water) at a maximum flow rate

of ten drops/minute. At this state, the amino acids were.

tightly held near the top of the column. The column was
rinsed with 100 ml distilled water to wash out the last

traces of anionic and neutral materials* The amino acids

were then eluted with NH40H (72). The first elution was
with 0.15 N NHIOH (50 ml/ml.of resin bed) and then with 2 N

NH40H (25 ml/ml of resin). Elution was also carried out in

the cold (column was placed in ice and water). The NH40H

elute was collected and the column was washed with distilled

water until the eluate was ammonia free (phenolphthalein

test). The combined eluates were collected and concentrated

under vacuum to an appropriate volume (usually 1 ml/l gm

fresh tissue). The purified amino acids obtained in this
way were adjusted to pH 2.25 and were stored in a freezer

(-15 C.). The resin column was regenerated for the next
run by washing with 3 N HC1 (6 ml/HC1/1 ml resin bed).

E. Qualitative identification of amino acids in the extracts

Two-dimensional paper chromatography was used to
separate and identify the amino acids. A sample of 100 ul
of plant extract prepared as described in section D was

placed as a single small spot in the corner (2.5 cm from

each of the two edges) of a 30 x 40 cm piece of Whatman #3

paper. The spotted paper was developed in a chromatographic
_, -









chamber, which was maintained at' apprdzimately 250 C., in an
air-conditioned, room., The solvent used ,yere: butanol,
acetio acid and H20 for itbe first phase nd phenol-water,
for the second phase (71Q75).. Preparation of solvents: 1)
125 ml butanol plus 1255ml water plus 30 ml acetic acid
(lower phase discarded) upper phase was used for paper
chromatography, 150 ml of solvent were used in one trough
for two papers; 2) 160 ml of pure phenol and 40 ml of dis-
tilled water plus 0.5 ml of ammonia. Equilibration time of
papers and solvents in chromatographic chamber was always
two hours. A 1 per cent solution of ninhydrin in acetone
was used to stain amino acid spots. Pkper chromatograms of
known amino acids were first prepared, and then they were
used to compare with those of the plant extract chromatogram
to identify the-kind and number of the soluble amino acids
present in the extract. The colored spots were eluted with
10 ml 50 per cent ETOR and the optical density was deter-
mined spectrophotometrically (72,73,74).

F. Quantitative determination of soluble amino acids in pea
extracts

After repeated.tests, we rejected paper chromatography
as a quantitative method for amino acids because the method
did not yield consistent results. The equipment and tech-
nique available to us allowed only a qualitative or rough
quantitative measure of the amino acids.




*+' ' , '' + ' + ; .'4 : 1 .

, 27

For quantitative separation and measurement of the

amino acids in bur extracts, we used an adaptation of the
ion-exchange separation method of Moore, Spackman and Stein

(58).
1. Preparation of ion-exchange columns.-The acidic

and neutral amino acids were separated in a column of cation

exchange resin Aminex-MS IR-120 Fraction D, particle size

56 + 9 u.* The resin bed was 150 cm long. The basic amino
acids were.separated on another column of Aminex-MS,

Fraction 0, particle size 40 + 7 u.* The resin bed was 15 cmi

long. The temperature in both columns was kept constant by

the presence of a watei jacket around the columns through

which water of the desired temperature was circulated.

Elution was accomplished by passing solutions of sodium
citrate buffer o, appropriate pH values through the columns.

For separation of the acid and neutral amino acids,
a thick walled c6hromdtographic column (obtained from Sci-

entific Glass Apparatus Company) 0.9 cm inside diameter and

165 cm in 'length walib used. iA the bottom of the column was
a sintered plate to retain the resin. The resin was washed

with four times its volume of distlled water, then washed

twice with 0.2 N pH 3.25 citrate buffer. The resin was kept

in slurry form in the buffer until used. Packing of the long
I I I I .
SPurchased from BIO-Rad Laboratories, Richmond,
California.
,p .. ,

r,~ ~ ~~: 1,': ,:








column was done in five sections. Before the first section

of resin was poured the outlet of the column was closed.

Then a slurry containing one-fifth of the total resin in
buffer was added to the column. The amount used in this, and

in four subsequent additions, was 19.6 gm of resin in 60 ml
of buffer. After the slurry had been added, the column

*outlet was opened, and after 1 cm of resin bed was settled

under gravity flow, then air pressure (5.8 psi 30 cm
mercury) was applied at the top of the column. After the
resin settled to constant height, the supernatant extra

buffer was withdrawn and the next section was poured on top
.of the firm surface of the first section. The procedure was

then repeated for each additional section until all five

sections had been added and packed. The final height of
resin must be several centimeters more than 150 cm to allow
for further packing of resin during operation. (It was

161 cm in the beginning.) An extension tube was attached to

the top of the chromatographic column by means of rubber

tubing, and this in turn was connected to a separatory funnel
containing buffer. A pinch clamp was installed below the
funnel. Before the column was used it was treated with 100
ml of 0.2 N NaOH and then was-equilibrated with buffer of

pH 3.25. The rate of flow was 18 ml/hr. This flow rate was
maintained by applying an air pressure of 3.2 psi to the top

of the buffer reservoir (separatory funnel), during column




. i V '. a ?' -' , ... -; '
,, ... ": "'*" ''." . Si,*




operation (temperature during operation of long column was

49 + 1o C.).
For separation of the basic amino acids, a short
column (15 cm) was prepared.with 12 gm of Aminex-MS Resin
(Fraction 0, size of particle 40 + 7 u). The resin was
washed carefully several times with distilled water and then
three times with citrate buffer of pH 4.25. The column was
packed under five pounds pressure in two sections, in a
manner similar to the packing of the lbng column. During
operation, the ',olumn was usually under two pounds of air
pressure at 306 C., and it had a flow rate of 22 ml/hr..
This column was used. ozly,'for the basic amino acids, and it
took only nine hours for one ext;rat to be analyzed. Citrate
buffers of different pH' were prepared as shown in Table 2.
Thiodiglycol (TG) was added to the buffers before
use. Brij 35 detergent was used only once every four to
five weeks since it caused problems in work with the drop
counter. (Brij 35 reduces surface tension and thus the
volume of drops change.) It was necessary to wash the column
after Brij 35 addition for two to three days to eliminate
Brij 35 effect. In all the citrate buffers, phenol protected
the buffer against microbiological activity. A final adjust-
ment of pH of the buffers was necessary, usually 1 ml of 50
per cent NaOH or 2 ml of concentrated HC1 caused a change of
about 0.01 unit in pH value of the buffers (1 liter).

9' '





J


,1i


TABLE 2

Preparation of Buffers


Na Citric NaOH HC1 Water to a .
Conc. acid 97 % (cone.) final Vol. Phenol TG* Brij+
pH (normal) gm Fm ml of (liter) gm ml 35 ml

2.25 0.20 105 42 80 5 5

3.25 0.20 840 330 426 40 40 10 5

4.25 0.20 840 330 118 40 40 10 5

5.28 0.35 491 288 136 20 20 5

4.26 0.38 532 312 307 20 20 5


*TG (Thiodiglycol).

ABrij 35 detergent solution--prepared by dissolving 50 gm of Brij 35 in
150 ml hot H20.


ft ft I. I


1'~ P ;1


'. 2.; r:
J,







31

To prevent bubble formation when the temperature was
raised to 50 C. in the column, it was necessary to boil the
buffer. (Volume of buffer should be checked after boiling,
in order to keep Na concentration constant.) Hot buffer was
transferred to the separatory funnel (buffer reservoir) via
a long-necked funnel to introduce the boiled buffer below
the level of a paraffin oil layer constantly maintained in
the buffer reservoir. (There must always be a layer of oil
over the buffer to prevent 'air mixture with buffer.) Addi-
tional buffer for immediate use was kept under a layer of
paraffin oil. The buffer reservoir Was connected to the
top of the column via two male and female ball joints (a
small amount of vachum grease was always used to make tight
connection).
2. Operation of the long column (for neutral and
basic amino acids).-The load of the column should not be
very much more than the amount that was used for paper
chromatography, usually about 1.0 umole of each amino acid
was satisfactory. A sample of the purified plant extract
was applied on top of the resin bed with a bent tip pipette
(that is, it was carefully run down the glass wall of the
column in order not to disturb the surface of the resin bed).
The temperature of the column was brought to 500 C. before
the addition of sample,. then 3.2 pounds pressure was applied
until the top surface of the extract layer was brought to







32

the resin Surface in the column. Then, ml of buffer at
pH 2.25 was added and after this, buffer also was brought
level with the top of the resin bed. Then the column was
connected to the separatory funnel containing pH 3.25 buffer.
Air pressure of 3.25 psi was applied. Effluent was collected
in 1 ml fractions with a fraction.collector (Research
Specialties Co., Mod. 1205). After 260 tubes were collected,
buffer at pH 4.25 (0.2 N Na) was substituted for the buffer

at pH 3.25 to elute the neutral amino acids. Usually, in
the long column the time for a single extract analysis was
about 24 hours (up to the isoleucine and leucine peak).
To determine the location of different amino acids
in the effluent, a series of known amino acids was tried.
After the location of each amino acid was known, several
tests on synthetic mixtures were made to make sure of repro-
ductibility and also the percentages of recovery of amino
acids from the column. The order of appearance and degree
of separation of the peaks was completely reproducible, Re-
covery percentages for each amino acid were determined as
shown in Table 3. The, columnn must be renovated after each
run, since otherwise all basic amino acids will be retained
in the column. Renovation is accomplished simply by passing
100 ml of 0.2 N MNao through, he column. Subsequently, the
column must be washed thoroughly with the appropriate buffer
to become reajusted before being .reisd. There is no need

2* .' * *' .; ..
1V 2*
*2* :. .




: irr ".i. ", ".


TABLE 3

Percentage Recovery o A 6ino, Acids from the Long and Short Column


SPer Cent

1. Aspartic acid 105
2. Alanine 95
S*95

3. Arginine 89
S 4. 'Glutamit aci. 92
4. i; 92

5. Glycin, 79
0 C, '
6. Histidine 92

7. Isoleucine 92

i8. Leucine 93

9. Lysine 90

i10. Proline 86

11. Phenylalanine 98

12. Serine 103

13. Homoserine 95

14. Threonine 102

15. Tyrosine e 98

16. Valine 98


'A'
* 0' .












for pressure during renovation of,the column and readjustment.
It is good to add 1 ml Brij 35 solution to the 100 ml NaOH
to help clean the column. (However, this does not need to
be done more than once a month.)

3. Operation of short column (for basic amino acids).-
The general procedure is the same as that with the long
column, but column loading with amino acids should be less
than with the long column. Air pressure of two pounds is
enough to produce a 25 ml/hr flow rate. The temperature
used was 32 + 1 0. Elution was with citrate buffers at pH
4.25 and 5.28 (.35 N Na) (100 tube with pH 4.25 then pH
changed to 5.28). The short column also has excellent repro-
ductibility, and recovery is more than 95 per cent for all
amino acids.
4. Colorimetric analysis for the amino acids (63).-
The reagents necessary for color development are prepared
as follows:: 1) stock NaCN 0.01 M. (49 mg, per cent);.2)
acetate buffer 3 N pH 5.3 270 gm Na acetate (NaCH3COO, 3 H20)
plus 50.ml concentrated glacial acetic acid and 200 ml dis-
tilled water brought to 750 ml with distilled water. This
buffer is Very concentrated (3 N) and-does not need any
microbial protecting agent; 3) acetate cyanide, 2 ml of
solution one and 98 n:l acetate buffer; 4) ninhydrin solution,
3 per cent ninhydrin in methyl cellosolve (ethylene glycol
monomethyl ether). Methyl cellosolve was checked each time



'J A
14\ ;,






S. ... 35

before use to make sure 'it was very low in peroxides. To
2 ml of methyl celiod1-Ve 6in a test tube, 1 ml 4 per cent KI
was added, if it,wt s colorless or a very light straw color,
the methyl cellosolve was regarded as satisfactory, other-
wise it needed to be dis1il4ed; 5) dilgnt: 50 per cent
isopropyl or ethyl alcohol in H20.
Test tubes collbcted. in the fraction collector were
tested for amino acids by addition of 0.5 ml acetate-cyanide
(reagent 3) and 0.5 ml ninhydrin (reagent 4) to each tube.
The contents of the tubes were thoroughly mixed by shaking
and swirling. The tubes then were placed in boiling water
bath (in a rack of 50 tubes) and were allowed to remain
there for 15 minutes. The heat supply to the water bath
was such that boiling started again shortly after the rack
of tubes was placed in the water bath. After 15 minutes,
the tubes were taken out of the water bath, and the contents
of each were diluted immediately with 5 ml of 50 per cent
EtOH (at room temperature). The tubes were then shaken to
mix the solutions. After half an hour, allowed for color
development, the optical density (0,D.) was read at 570 mu
in a Spectronic 20 colorimeter for all amino acids. For
proline, the tubes were also read at 440 mu. All readings
were against a blank of 50 per cent alcohol. An additional
5 or 10 ml of diluent were added to tubes with color in-
tensity above 0.80 O.D. In such cases the resulting 0.D.









values were multiplied by 1.7 (7 ml + 5 ml/7 ml) or 2.4

(7 ml + 10 ml/7 ml) for correct amino acid concentration.
The reagents should not be mixed together unless they are

going to be used immediately, since the resultant solution

is unstable and is useless after two hours. The reaction

mixture taken from hot water was immediately diluted because

pausing for cooling at this step before addition of diluent

alcohol produced high background values. The stream of

diluent was always directed at the center of the test tube,
to introduce a maximum of air. The diluted reaction mixture

should be allowed to cool to room temperature before reading
in the Spectronic 20. The color produced is quite stable.

After 12 hours there was only a 10 per cent decrease in the

color intensities. A standard solution of. leucine was always
tested with each run to make sure of the efficiency of the

reagents.
Color intensities of individual amino acids were

plotted against tube number. The area enclosed by the re-
sulting curves was a direct measure of the total amount of
the amino acid.

Test tubes for this *ork must be well matched for

light transmission. However, commercially matched tubes

were not available so ordinary tubes were examined, and a

large matched set was collected.




* I' r ;


37

Washing of test tubes is important. Used tubes were

first rinsed with hot water and then were washed with Fisher

Sparkleen detergent in hot water. They were again rinsed in

hot water and finally were rinsed two times in distilled H20

and were dried in an oven at 1100 C. Occasionally the tubes

were also washed in a dilute (0.2 N) solution of hydrochloric

acid and were then rinsed in distilled water.

G. Protein determination

For the measurement of total soluble protein, the

handling of the plant material for sampling was the same as

for the amino acids. Samples for protein determination were

prepared in the following steps:

1. A weighed amount of leaves (1 to 5 gm) were rinsed

with cold distilled water.for a short time..

2. These leaves were homogenized in ice cold condition

with a hand tissue homogenizer (in 25 ml distilled

H20/gm leaves) for five minutes.,
3. The well homogenized tissue was quickly passed

through cheesecloth (ice cold condition). Filtrate

was brought to a measured volume at about 25 ml/gm
tissue.

4. Equal amounts of ice cold 5 per cent trichloroacetic
acid were added to',the filtrate in number 3 above,

with shaking, to. obtin complete precipitation of

the protein.

\* *;., ." "**1 t >







38

5. The sample was centrifuged for.ten minutes at
lOOO.X
1o000 G.
6. The precipitate was promptly Washed with acetone to
remove chlorophyll and other pigments (20 ml acetone
per gm of tissue).
7. After another ten minutes centrifugation at 1000 G,
the supernatant pigment was removed and saved for
total chlorophyll. determination.
8. The precipitate, which was mainly protein, was dis-
solved in 0.91 N NaOH (40 ml NaOH/per 1 gm tissue)
for protein estimation by the phenol reagent (49).

9. A measured volume of the protein solution (usually
0.25 to 0.5 ml) was placed in a graduated 10 ml test
tube, and 5 ml of a mixture of the following reagent
was added to the tube:
Reagent A 100 ml 2 per cent NaCO in 1 N NaOH
Reagent B 1 ml 4 per cent Na tartrate in H20
Reagent C 1ml2 per cent CuSO4, 5 H20 in H20.
(The three reagents must be mixed together shortly
before use.) After addition of the reagent the tube
was shaken vigorously and was left at room temperature
for ten minutes.
10. A volume of .5 ml of Phenol reagent (Folin-Ciocalteu)
was added to the sample tube; the tube was shaken
well and was allowed to stand 30 minutes at room
temperature.




,, ?,, 1 l' l .i*P; a! ; r i" I r ,


39

11. Prexraxti on. ast andardi curve:
j ,A ' . .

A standard curve was prepared using a solution of

bovine serum albumin (amounts ranged from 50 ugm

through 500 ugm). The standard curve obtained did

not follow Beer's law for high concentrations, so

the sample must be low in concentration in order to

get an accurate reading. It is very important to be
careful not to denature protein during handling;

dilute bovine solution denatures usually very fast.

The colored solutions obtained were read in a
Beckman D.U. spectrophotometer or Spectronic 20 at

wavelength of 750 mu. The values presented are

usually an average of two or three determinations.
For determination of total protein we used the method
of Lowry (50). This methodss based on the final color

formation due to both the Biuret reaction and the Polin
reagent (reduction of the phosphomolyhdic-phosphotungstic

reagent by the tyrosine and tryptophan in the treated pro-
tein). There are two disadvantages to this method: 1) the

amount of color yield varies with different proteins. There-

fore, it is good'only for comparison of the quantity of two

proteins of the same nature; 2) the color is not strictly
proportional to concentration since for high concentration
it does not completely obey Beer's law; thus dilute solutions

of protein should be used. Since the color intensity per

unit of protein varies wit$h different proteins, there should

;, ,. .. ^ 'y '




** (I *' ::. :-, .;. ;


1: *" :0

be a correction factor to calculate the absolute amount of
the protein in comparison to a standard such as bovine serum
albumin. .This is .when the absolute quantity of a protein
needed to be determined, otherwise tor determination of the
relative protein contest of the same plant variety under
two different growth conditions there is no need for such a
factor to be applied. In the research reported here we
were more interested in differences (relative values)
rather than the absolute quantity. Despite the disadvantages
of the Lowry method, there were several advantages, which led
us to adopt it. The method is more sensitive and convenient
than is the digestion and Nesslerization method. Also, it
is 10-20 times more sensitive than the U. V. absorption
method of protein determination. It is much less liable to
inaccuracy due to turbidity, while it is almost 100 times
more sensitive than the Biuret method for low concentrations
of proteins.

H. Chemical treatment techniques

Metabolites were applied as a leaf spray to plants
under different temperature conditions. Plants were sprayed
every other day until dripping wet. For a better wetting of
leaves, surfactant (Tween 20 -0.01 per cent) was added to
the metabolite solution. The different metabolite solutions
which were the same as those employed by ietellapper (32),
included:







41

1. Vitamin B mixture.

2. Vitamin C.

3. Nucleosides.

4. Sucrose.

The composition of chemicals metabolitess) sprayed on leaves

is shown in Table 4. The control plants were sprayed with

water plus surfactant. Chemical treatment continued for

several weeks. Plants under both optimum and high tempera-

ture condition were treated with these metabolites.

Measurement of height, dry weight, fresh weight and finally

flower and fruit formation was carried out to examine the

effect of these metabolites.



























Si ' i :
.;; J* II "i







4.2


o t o TABLE 4
Composition of Metabolite Solutions Sprayed on Leaves

Composition of Metabolite Solutions Sprayed on Leaves


Concentration


Mixture

1) Vitamin B Mixture

Thiamin

Riboflavin

Ca-pentathenate

Biotin

Pyridaxin

Nicotinic acid

SInositol

Folic acid


2) Riboside Mixture

Adenosine

Guanosine


3) Ascorbic Acid


4) Sucrose Solution

Sucrose

Sulfanilamide


mg/1

mg/l

mg/1

mg/I

mg/1

mg/1

mg/1

mg/1


x 10-4 M

x 10-4 M


1 g/l




1 g/1

125 mg/1


41


20

5

20

1

20

40

200

5




. : . .- .. . . .







SAMPLING AND PHYSIOLOGICAL AGE


Sampling from plants under different environmental
conditions according to their chronological age is not
advisable when studying the biochemical effects of one of
the environmental variables. Differences in physiological
age at a given chronological age soon develop, introducing
a second variable which may have pronounced interfering
effect on the study of the first variable.

In the experiments with high and low temperature
effects, after a short time plants of the same chronologi-

cal age would not have the same physiological age, since
time of flowering and fruit formation would be profoundly
affected by the temperature. Thus, there are biochemical
changes associated with the stage of maturity while it is
biochemical changes due to temperature, at equivalent
*, . ..






Seeds, an then.the YoAng seedlings, were hand-graded
to obtain uifp i sze, n lants ,by aysarding unusually small

or large ia Samples i for ext action were taken from at
... .. a .'. ,* .. .'. '" '* ', "' ... '




*,'I. K



44

least five plants. The five plants were chosen from a rather
large number of plants (usually 60) in a random way (67).
To eliminate the problem of physiological age dif-
ferences, a numerical index of vegetative development, the
plastochron index of Erickson (12), was determined in each
case and samples were taken for analysis at equivalent
indices. The period between the initiation of successive
leaves as well as the size of leaves are features considered
in the plastochron concept. A plastochron is defined by
Erickson as the time between corresponding stages of develop-
ment of successive leaves. (For a corresponding stage of
development, it is possible to choose either the initiation,
maturation or attainment of a standard length of leaves.)
In our research, which was carried out in climatic controlled
chambers, the physiological ages of plants grown at high
and optimum temperatures were calculated according to
Erickson's formula (12,13,53,56).

log L(a) log20
P.I. = n + log L(a) log L(a+l)

where::
n a the serial number of leaves which are equal to
or just larger than an arbitrary value, which
is 20 ,.m in our experiment,
L(a) is the length' of the highest leaf with length
equal to of larger than 20 mm,

S j. .: ? 1 .* i "- k ,'









L(a+l) is the length of the next higher leaf smaller
than 20 mm in length,
P.I. = plastochron index.
Usually the P.I. is a good index of the developmental age for
a plant which has stable spiral phylotaxy (12). During the
vegetative period of growth, the P.I. is linearly related to
time. Plotting of P.I. values of each day against time after
germination gives almost a straight line and can be used as
a good substitute for chronological age. If the length of
the leaf a is exactly equal to the arbitrary length value,
the plant would have a P.I. equal to n, but this does not
happen frequently; usually L(a) is more or less than that
value. For example, if L(a) equals 25 mm and L(a+l) equals
17 mm at a given time, it is clear that the P.I.: of the plant
is more than n and less than (n+l). In practice, the P.I.
calculation is carried out by counting all the leaves that
are equal to or larger than 20 mm. Then measurements are
made of the length of two successive leaves which respectively
are less L(a+1) and just equal to or more than 20 mm (L(a)).
Therefore, in our example, if n = 15, L(a) 25 mm, and
L(a+l) = 17 mm, the P.I. value would be:.

25
P1 log 25 log 20 log 20 1
P.I. 15 + log 2 log 2 1558
log 7









All samples taken for extraction from plants grown

in the two temperature conditions were taken at such times

as to have the same P.I. value + 0.2 P.I. units.

After the plants passed the vegetative period and

reached the reproductive stage of life the plastochron index

could not be used any more. For the extracts taken after

flowering started, the time after flower initiation was

considered only as the chronological time. Three extracts

were taken during the-vegetative period, one in the flowering

stage and one seven days after flowering when small fruits

are formed. In most cases, plants under high temperature

conditions did not produce any fruit or at least not any

perfect fruit, so they remain for a long time in the vegeta-

tive stage and ultimately start rosette type of growth. It

is evident that biochemical comparison of this group with

the optimum temperature group (which have fruit and are in

a perfect reproductive stage) is of little value. That is

why further samples were not taken. Fruit formation in one

series of plants under optimum condition was inhibited simply

by the removal of pistil or ovary with a sharp needle to

produce similar physiological conditions in the optimum and

high temperature groups. Growth characteristics of this
group were compared with that of the high temperature group.












RESULTS AND DISCUSSION


I. Growth Characteristics of Plants Under Optimum
and High Temperature Conditions

A. Elongation (increase in total height)

The pattern of growth (height increase) in the rela-

tively heat-sensitive pea variety Greater Progress was quite

different at high and optimum temperature conditions (Figures
1 and 2). The plants grown at high temperature grew very

rapidly during the first weeks after germination (14 to 19

days after seeds were planted). At optimum temperature, the

rate of elongation gradually rose to a maximum at about 19

days after seeds were planted. The sudden and sharp drop

in the rate of elongation at the high and optimum temperature

condition coincided approximately with the time that the

reserve materials of the seed were exhausted. However, the

drop in growth at this stage was greater at high than at

optimum temperature. Flower bud formation usually started
about 25 days after planting at the optimum condition and

occurred about three days earlier under the high temperature

regime. Flowering caused a decline in the rate of stem

elongation, and by the time fruit set started, the elongation

stopped altogether. At the high temperature condition there

47




.1 (


Fig. 1. Daily height increase of pea plants under two tempera-
ture regimes.
(a) early drop in elongation
(b) flowering period
(c) fruit formation occurring
(d) abscission of flowers and immature fruits occurring at
the high temperature condition
(e) second flowering period at high temperature condition
(f) second absicssion of fruits and flowers





HEIGHT INCREASE (MM./DAY) ,

I I I


0o-

0'


00-3 ,


\
00
' /


/
0 0L


Ns


0

I





O
0





FIG. I


0Ih


0

-< r
C)
> ro
n1l .4
-I
m


0







-0
rn






























Fig. 2. Growth (height increase) of the pea plants under high
and optimum temperature conditions as compared with the groups of plants
which were depistillated (in order to stop fruit formation) at the
optimum temperature condition.












-0- OPTIMUM TEMPERATURE
0 HIGH TEMPERATURE
-- OPTIMUM (PISTIL REMOVED)


o--o

JO


15 20 25 30 35 40 45


DAYS
DAYS


FIG. 2


cm 25-


20-


__ _


.0---0







52

was a decline in the rate of height increase during the

flowering period similar to the optimum temperature condi-

tion, but the elongation never ceased altogether because
there was no perfect fruit set at this condition. At high

temperature, abscission of the flowers usually occurred;

if not, any young fruit that did form abscissed very early

in the developmental stage. After the first flowering period

stopped without any fruit being formed, the rate of stem

elongation of the plant under the high temperature regime

again increased. This increase in elongation continued until

a second flowering period started. Since we were interested

in studying the effects of temperature per se rather than a

possible secondary effect of flower initiation and abscission,

we attempted to produce more comparable plant material by

removing the pistils of flowers as they formed in a group of

plants at optimum temperature.

The pattern of. stem elongation seen after flower

initiation in the high temperature series could be obtained

also under the optimum temperature condition when the flowers

were depistillated.in order to stop fruit formation (Figure 3).

The main difference in the pattern of growth between the two

conditions seen in this experiment was that flowering

occurred more often and much faster when the pistil was re-

moved by hand at optimum temperature than when the flower

and fruit drop was caused by high temperature. The increase



























Fig.
tions (pistil
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)


3. Daily height increase at optimum temperature condi-
removed).
early drop in elongation
flowering period
fruit set
pistil (or very small fruit) removed
flowering started for second time
removal of pistil or young fruit
flowering started for third time
removal of pistil or young fruit






HEIGHT INCREASE
0


(MM./DAY)

0 -


FIG. 3


O






n




m
M -g







M o0
m

C ro






z
O 4


rl
0)



0-


CM-







ro
r-0


--


--'---


I I









and decrease in growth rate was also sharper and more

frequent in the depistillated, optimum-temperature series.

The life span of plants under optimum conditions usually

was about two months, while at high temperature it usually

exceeded three months.

To test whether the inhibition of fruit formation,

and hence longer period of growth might be due to a failure

of pollination, groups of plants grown under the high

temperature condition were brought to optimum-temperature

for one week during the flowering period. There was no

significant improvement as far as formation of perfect fruit

was concerned. However, if they were kept for a period

longer than one week at optimum-temperature, the fruit set

occurred in a normal way. These observations eliminate the

possibility that inhibition of fruit formation was due to

the simple failure of pollination at high temperature. How-

ever, this does not indicate whether pollen was viable or

not at high temperature. Of course, high temperatures might

have affected the rate of maturation of flower pistils and

stamen so that they did not mature at the same time, a

process that is likely to happen in many self-pollinating

plants under certain environmental conditions. If the

failure of fruit set at high temperature was due to deranged

maturation of flower parts directly caused by the elevated

temperature, 3:;t would seem obvious that the initiation of

this derangement occurred more than a week prior to pollina-

tion.










A number of the growth studies were repeated with a

more heat-resistant variety of pea (var. Wanda) at the same

time and under the same growing condition. In contrast to

the results with the heat-sensitive variety (var. Greater

Progress), there was not very much difference in the growth

characteristics of the var. Wanda under the two conditions.

Although the number of flowers and total yield was less at

high temperature, there was no problem of flowering and

fruit formation as observed in the case of the heat-sensi-

tive variety. (The data for this part are not presented

in this dissertation.)

B. Fresh weight increase at high and optimum temperature

During the first three weeks after planting the seeds,

there was little difference in amount or rate of increase in

fresh weight at the two temperature conditions (Figure 4).

However, three weeks after the seeds were planted the rate

of fresh weight increase dipped sharply in the high tempera-

ture group but not until the fourth week in the plants which

were at optimum-temperature. Later on, after the fifth week

of growth, there was an increase in the rate of fresh weight

accumulation at the high temperature condition. This in-

crease occurred after the first abscission of flowers, at

the time that rate of growth and elongation was increasing

at high temperature, while at optimum-temperature the rate

continued to decrease. However, increase in fresh weight vs.

































Fig. 4. Total fresh weight increase and the weekly increase
of fresh weight at high and optimum temperature conditions.




.r.
"!i: "~"; "" ';" ~'
'd 1'El ;'.,":
CI$C(~
i


12


II -


10-


9


8




I-
LL
rt
5


4


3-


2


1 .9


-0- OPTIMUM TEMPERATURE
-0- HIGH TEMPERATURE






<"


/
-G-0,
^ "1


- -,O


I I I I I


[ OPTIMUM TEMPERATURE
- HIGH TEMPERATURE


2 3 4 5

WEEKS AFTER SEEDS WERE PLANTED
,c 1 f : :


FIG.4
*/, v ^ ., ,. 1-" '


' 58


A




/ ''*' .. ;" ;;





time had a lower rate at 'high temperature, and the total

fresh weight was much lower,iat high temperature; part of

this decrease rate of fresh weight accumulation at high

temperature seems to be due to the abscission of flowers and

young fruits which could cause a lowered rate of increase in

total fresh weight.

C. Increase in total dry weight at high and optimum-

temperature

Total dry weight per plant as well as weekly increase

in dry weight were almost the same at the two temperature

conditions during the first three weeks of growth, but later

the rate of dry weight increase was much faster at optimum

conditions (Figure 5). After the fifth week of growth, the

rate of dry weight increase dropped at high and optimum

conditions simultaneously. However, the rate of increase

of dry weight continued to drop at optimum conditions while

at high temperature it started to increase again after the

sixth week of growth. Here again, this increase was mainly

due to rapid vegetative growth that started after the first
abscission of flowers and fruits occurred at high temperature

conditions. The final dry weight per plant (after seven

weeks of growth) was almost twice as much at optimum-

temperature conditions as at high temperature.

































Fig. 5. Total dry weight increase and the weekly increase
of dry weight at high and optimum temperature Conditions.





61
2.5 -
--' OPTIMUM TEMPERATURE
2.25- - HIGH TEMPERATURE


2.0-


1.75-


I- 1.5 -
:...., :. . .o




01.0



.
.75




.25- o










SLi
0L ..-------
.5- OPTIMUM TEMPERATURE ,. *
B Q~1 HIGH TEMPERATURE




- .






o 2 3 6 .7

.WEEKS AFTER SEEDS WERE PLANTED


FIG.5









D. Increase in percent of dry weight


The percent of dry weight was almost the same at the

two conditions, and there was a continuous rise in the

percentage of dry weight as the plants became older. After

fruit set, however, there was a very rapid rise in the

percent dry weight at optimum-temperature conditions, while

at high temperature there was a slow rise in percent dry

weight.

E. Shoot-root ratio

The ratio of shoot weight to root weight was almost

the same through three weeks of growth at the two tempera-

ture conditions (Figure 7). Later the ratio increased at

the optimum condition (as fruit formation started) and be-

came much more than at the high temperature condition. The

low ratio of shoot to root at this period, in plants grown

at high temperature, was partly due to abscission of flowers

and fruits. But, after the fifth week of growth, the ratio

in the high temperature group started to increase; and

finally, by the sixth week the ratio became equal to that

of optimum conditions. This increase in shoot-root ratio

probably was a result of the spurt in vegetative growth

observed at this time in the plants exposed to high

temperatures.














Fig. 6. Increase in per cent dry weight at different stages
of growth at optimum and high temperature conditions.
(a) Fruit set occurred in plants grown at optimum temperature
conditions, but not in plants grown at high temperature.


Fig. 7. Shoot-root weight ratio versus time.
(a) Ratio increases faster at optimum condition during
flowering and early fruit formation period.
(b) Ratio became higher because of fruit formation at the
optimum condition. Finally, fast vegetative growth after
the first abscission of flowers and fruit at the high
temperature condition increases the ratio.


f





20-

S18-
(9
LU
16-


14-
z

12
LU- .
o 10
z

| 4-


-0- OPTIMUM TEMPERATURE
-0- HIGH TEMPERATURE













O. 1;/


a -
J,


" 00


6









6


b/







]/ ,O

a0O
-0-





-0- OPTIMUM TEMPERATURE
-0 HIGH TEMPERATURE


FIG. 6and7


0


4- .


/-


i I p i I
S2 3 .4 5 6 7
WEEKS AFTER SEEDS WERE PLANTED


F I I


0.1 ,


i


i










II. Amino Acid Analyses of Peas Under Optimum

and High Temperature Conditions

A. General

The results of the quantitative analyses with ion-

exchange column chromatography of the plant extract for

their amino acids are shown in Figure 18 and in Tables 5

through 10. Results are expressed in terms of micromoles

of amino acid per gram fresh weight. Values for the amino

acid quantity present were calculated on the basis of the

color yield with ninhydrin of a standard solution of

leucine, observed in frequently repeated standard curve

determinations. The results in most cases were repeated

two or three times to make sure of the reproducibility and

consistency of the location of the peaks for each amino

acid in the effluent. After reproducibility and consistency

of the chromatograms were well established, further analyses

of the extracts were carried out only once. After several

analyses, it was found that elaborate purification of the

plant extracts was not necessary in order to make quantita-

tive determination of amino acids. (Efficiency of the column

for analyses of amino acids in the partially purified extracts

was almost the same as for well-purified extracts.)








66

B. Problems in separation and identification of individual

amino acids

There was some uncertainty concerning the location

or adequacy of resolution of certain peaks in the chromato-

grams. Careful control of conditions, or additional steps

resolved a number of these problems. For example, the

location of valine, isoleucine and leucine peaks depended

on the time when citrate buffer of pH 3.25 was replaced

with the citrate buffer of pH 4.25.

The asparagine and glutamine peaks normally were

combined with each other and serine, making it impossible

to determine the quantity of these amino acids. Therefore,

determination of the quantity of asparagine, glutamine and

serine was carried out by the hydrolysis of the asparagine

and glutamine in the extract. For this purpose, the puri-

fied plant extracts were hydrolyzed in a solution of 0.1 N

HC1 at 1000 0. for three hours (20 ml HC1 for 0.5 ml extract

equivalent of 0.5 gm fresh tissue); the hydrolyzed extracts

were chromatographed, and the increase in quantity of

aspartic and glutamic acid as compared with that of a non-

hydrolyzed extract was determined and calculated as

asparagine and glutamine. Serine determination after

hydrolysis was also carried out. In all cases, the amount

of asparagine was much more than glutamine. Synthetic

mixtures of a known quantity of serine, asparagine and
|A |







67

glutamine were hydrolyzed in the same way and then chromato-
graphed; the results obtained showed that there was an 8
per cent loss due to hydrolysis and handling of the sample.
Therefore, values in the hydrolyzed extracts were corrected'
for this loss. In analyses of extracts, it was observed
that a number of peaks appeared early before the aspartic
acid, which, on the basis of its acidity, one would expect
to find as the first peak. These early peaks were found to

be mainly peptides, soluble in 80 per cent ethanol. Treat-
ment of extracts with 0.1 N HC1 caused the disappearance of
all of these peaks except one. The peak immediately before
the aspartic acid (the one that did not disappear on
hydrolysis) appeared to be methionine sulfoxide, according
to the location of a known sample of methionine sulfoxide
in a chromatogram of a synthetic mixture of amino acids.
Assignment of methionine sulfoxide to this peak is consistent
with the results of Lawrence (43) in similar analyses.
Methionine is well known as an unstable amino acid that
oxidizes readily to methionine sulfoxide during handling and
storage of plant extract. A hydrolysis method was also
carried out for exact determination of the quantity of
methionine sulfoxide, because the peak of methionine sulf-
oxide in unhydrolyzed samples coincided with one of the most

abundant alcohol-soluble ,peptides of pea seedlings, known as

-glutamyl alanine (74). Finally, the total amount of







68

peptides was calculated by summing all the peaks appearing

before aspartic acid (minus methionine sulfoxide). Proline

determination was not satisfactory in most cases. Usually

the large size of the glutamic acid peak, which appeared

just before the proline peak, caused the two of them to
overlap. The low optical density yield of proline per

umole (even at 440 mu wavelength) with ninhydrin reagent

and also the low content of proline in the extract, made

proline contamination an insignificant factor in the glutamic

acid analysis. However, in most cases these same factors

made it impossible to obtain quantitative data for proline

in the extracts. There was one peak (designated as unknown

A) which emerged immediately after.proline and before

glycine. This peak became larger in the high temperature

condition during the flowering period. We did not have

sufficient material to carry out tests to establish the

identity of the substance giving this peak. However, the

peak is in the position to be expected for a-aminoadipic acid,

known to be a constituent of pea plants (43,74). The glycine

peak usually was low and coincided in part with some very

small interfering peaks. The values presented for glycine

are the average of two determinations on each sample. It

was necessary to use a certain amount of judgment as to what

constituted the true shape of the glycine curve. Accordingly,

the precision and accuracy is not as good for glycine as for

most of the other amino acids.









0. Weekly changes in amino acid content at high and
optimum temperatures
1. First period of sampling.-Results are presented in
Table 5 and Figure l3for the total alcohol soluble (free)
amino acids content of leaves (um/gm f. wt.) of peas, one
week after seeds were planted (approximately three days
after germination, when no fully expanded leaves were present).
The amount of total free amino acid in leaves of the high
temperature plants was much greater (1.7 times) than in the
optimum temperature plants. The amount of each individual
amino acid is shown in Table 5. This early time in the life
of the pea plant, one should remember, is a period of active
hydrolysis and utilization of reserve proteins of the seed,
as well as a period of synthesis of new proteins in the
developing seedling. Not unexpectedly, amides were present
in very high amounts at both temperature conditions. How-
ever, the total amides asparaginee plus glutamine) at high
temperature were almost 2.5 times higher than at the optimum
temperature condition. More amino acid and especially more
amide formation at high temperature could possibly be due to
more rapid protein breakdown and faster growth at high
temperature during the first week of growth. The total pep-

tides content, homoserine and also methionine sulfoxide, in
both extracts were higher in the first week than at any other
time. In contrast, the amount of isoleucine and leucine was












TABLE 5

Amino Acid Content of Leaves First Period of Sampling

pM per gram fresh weight
Temperature
Amino acid Optimum High Ratio

Methionine sulfoxide 2.72 3.24 1.20

Aspartic acid 2.96 2.04 0.69

Serine 2.85 3.26 1.14

Asparagine 5.00 15.9 3.18

Glutamine 2.10 6.34 3.01

Threonine 0.34 0.47 1.38

Homoserine 1.43 4.23 2.95

Glutamic acid 3.27 4.95 1.51

Unknown A 0.61 0.82 1.34

Glycine 0.96 0.48 0.50

Alanine 1.82 2.12 1.16

Valine 0.77 0.98 1.27

Leucine Trace Trace

Isoleucine Trace Trace --

Peptide 5.60 6.20 1.11









low in the first week of growth. There was not a sufficient

amount of extract from these young and small plants to permit

measurements of the basic amino acids.

2. Second period of sampling.-Samples were taken from

a group of plants 14 days after planting at optimum and 13

days at high temperature (with P.I. values very close to

each other).

a. Amino acid of leaves. The amount of amino

acid in the extract of high temperature plants was almost

three times higher than optimum temperature plants. However,

at both conditions the amount of amino acids was less than

the previous sample which was taken about a week earlier.

Comparison of the individual amino acids in the

second sampling period showed that the most outstanding dif-

ference between the two extracts was the presence of a

rather large amount of amides in the high temperature

plants, while the amide content of the optimum temperature

plants was quite low. If amides were not included in the

total amino acid content of the high temperature plants,

then the total amount of amino acids in the high temperature

plants would be two times higher than at optimum temperature

conditions. The high amide content at the high temperature

condition could have been due to further proteolysis and

metabolism of seed proteins (which caused amide formation as

an ammonia detoxifying mechanism). On the other hand, the










high amount of amides and total amino acids could have re-

sulted from a lower protein synthesis at the high tempera-

ture condition, resulting in a larger amino acid pool (1).

Methionine sulfoxide and peptide content at high

temperature was also much higher than at the optimum

temperature. In general, all the other amino acids at high

temperature were in a substantially higher amount. The peak

of unknown A was very low (trace) in extracts of plants

grown under optimum conditions while it was high in the high

temperature extract. The results of analyses for this

period of sampling are shown in Table 6 and Figure 8.

b. Amino acid content of roots. Since there was

a very large difference in the total amount of amino acids

of leaves at the two conditions in the second period samples,

an extract from the roots at this stage of growth was

analyzed, to study the differences in amino acids in the

other parts of the plants as compared with leaves (Table 7

and Figure 9). Like the leaves, total amino acid content

of roots was higher (about 2.5 times) at high temperature

than at optimum temperature. In general, however, the

pattern of amino acids in roots was somewhat different from

that observed in leaves of the same plants. At both tempera-

tures the amount of homoserine was much higher in roots,

although it was almost three times higher in the extract from

plants grown at high than at optimum temperature. Homoserine









TABLE 6

Amino Acid Content of Leaves Second Period of Sampling


Amino acid

Methionine sulfoxide

Aspartic acid

Threonine

Serine

Asparagine

Glutamine

Homoserine

Glutamic acid'

Proline

Unknown A

Glycine

Alanine

Valine

Isoleucine

Leucine

Tyrosine plus
phenylalanine

b-aminobutyric acid

Lysine

Histidine

Arginine

Peptides


I


pM per gram fresh
Temperature
Optimum

0.08

2.22

0.25

0.80

1.10

0.55

0.81

1.33

Present

Trace

0.13

0.90

0.27

0.23

0.13


0.65

0.33

0.48

0.96

0.33

Trace


weight

High

2.05

3.52

0.49

2.30

6.54

2.82

1.57

3.83

Trace

0.29

0.14

1.90

0.63

0.32

0.48


0.51

0.97

0.24

1.37

0.84

1.50


Ratio

25.60

1.58

1.96

2.87

5.94

5.12

1.93

2.88


--

1.08

2.11

2.33

1.39

3.69


0.78

2.93

0.50

1.42

2.57



































Fig. 8. Chromatograms comparing amino acid constituents of
leaves of pea plants under optimum and high temperature conditions.
(Second period of sampling.) The upper chromatogram is from 0.5 gm
of leaves at optimum temperature, but the lower one is from 0.25 gm
of leaves at high temperature.



























,,\-!.'i '* '










18


1.6-


1.4-


. 2
tAsportic
Acid
1.0- Optimum Temperature


.8- Serine +
Asporogine and
Glutomine

.6- omoser

Mehionine Theoni Alonine Vline
4 Sulfoxide

SG 'no lsol 'ine Leucine
.2-


ZNNo Citrate Buffer, pH 3.2 2N NN Cirate Buffer DH 4
100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400
Effluent (ml)


High Temperature


Glutomic Acid


Alonine


FIG. 8












Amino Acid Content of


Amino acid

Methionine sulfoxide

Aspartic acid

Threonine

Serine

Asparagine

Glutamine

Homoserine

Glutamic acid

Proline

Unknown A

Glycine

Alanine

Valine

Isoleucine

Leucine

Tyrosine plus
phenylalanine

Y-aminobutyric acid

Lysine

Histidine


TABLE 7

Root Second Period of Sampling


Trace

0.16

Trace

0.25


0.11

0.41

Trace

0.51


pM per gram fresh weight
Temperature
Optimum High

1.17 2.05

0.69 1.13

0.37 0.57

0.58 1.12

1.47 3.20

1.01 2.05

11.37 30.12

0.77 1.07

Trace Trace

0.41 0.73

Trace 0.48

0.56 1.03

0.45 0.50

Trace Trace

Trace Trace


--

1.82

1.11



--




2.56



2.04
2.04


--


Ratio

1.75

1.63

1.54

1.93

2.17

2.02

2.66

1.39



1.78






























Fig. 9. Chromatograms of amino
optimum and high temperature conditions.
The upper chromatogram is from 0.5 gm of
but the lower one is from 0.3 gm of root


acid constituents of roots at
(Second period of sampling.)
root at optimum temperature,
at high temperature.












Serine
+Glutomine
+Asparagine









Methionine
Sulfoxlde

Asportic
Peptid Acid
Throoni


ty


Homoserine


Optimum Temperature


Glutomic Acid

Vaine



lsoleuclne Leucine


*--2-- 2N No Citrate Buffer. pH 3.25 .2N No Citrate


Buffer. pH 4.25 -


100 120 140
Effluent (ml)


160 180 200 220 240 260 280 3b0 320 340 360


Homorerine


High Temperature


Alanlne


Glutamic Acid
A


120 140 160 180


FIG. 9


1.4-

1.2-

1.0-




.6-

.4

.2-


380 400


__ __







79

is not a protein amino acid although it is known to be an
intermediate between aspartic acid and threonine. The high
homoserine content in roots of Leguminosae has been a subject
of considerable research and discussion by Virtanen and
Sasaoka (64,76). They showed homoserine is absent in seeds
but that it is synthesized during germination. The amide
content in roots also was much more (three times) at high
temperature than at the optimum temperature. Methionine
sulfoxide also was about two times higher than at optimum
temperature. It will be recalled that similar results for
amide and methionine sulfoxide content were observed in
leaves.

3. Third period of sampling.-This sample was taken
from plants 20 days after planting at high temperature and
22 days after planting at the optimum temperature condition
(with P.I. values very close to each other). The plants
were almost in their maximum vegetative growth and a few
days away from flowering. The total amino acids of leaves
from plants grown at high temperature were two-thirds of the

previous (13 days) sample, while at optimum conditions there
was a rise (by 1.4 times) in total amino acids. In this
sample, the total amino acids at high temperature were a
little more than at the optimum temperature (the ratio was
19:16). This situation, which was quite different from that
observed in the previous (second) sampling period, can be










interpreted as being due largely to two processes: 1) a

faster rate of respiration at the high temperature, which

would deplete the seed reserves more rapidly than at the

optimum temperature; 2) a faster rate of organic matter

production by photosynthesis at the optimum condition. The

photosynthetic system is probably more efficient in the

plants grown at optimum temperature (as the leaves are

larger and they have more chlorophyll). These factors

could have caused the increase in amino acids of the opti-

mum temperature plants, while at the same time, caused a

decrease since the previous sampling period in amino acid

content of plants at the high temperature condition. The

pattern of amino acids was more similar at the two tempera-

ture conditions in this sampling period than at any other

time. However, the unknown A was absent at the optimum

condition while it was quite a large peak at the high

temperature condition. Total amide content, also, was very

similar at the two temperature conditions. The results are

shown in Table 8 and Figure 10.

4. Fourth period of sampling.-Samples were taken when

the plants were in the full flowering stage (three days after

first flower bud formation). The high temperature plants

were 27 days post-planting while plants at the optimum con-

dition were 29 days. The P.I. values were nearly identical

for the two groups of plants. Total amino acids of leaves









TABLE 8

Amino Acid Content of Leaves Third Period of Sampling


Amino acid

Methionine sulfoxide

Aspartic acid

Threonine

Serine

Asparagine

Glutamine

Homoserine

Glutamic acid

Proline

Unknown A

Glycine

Alanine

Valine

Isoleucine

Leucine

Tyrosine plus
phenylalanine

I L-aminobutyric acid

Lysine

Histidine

Arginine

Peptides


0.20

0.49

0.21

0.10

Trace

0.69


0.35

0.51

0.23

0.20

0.35

0.88


II


pM per gram fresh weight
Temperature
Optimum High

0.45 1.02

1.94 2.53

0.41 0.50

2.00 1.66

2.09 2.21

0.94 1.19

0.97 1.34

3.17 4.13

Present Present

-- 0.31

Trace 0.18

2.24 1.58

0.37 0.50

0.23 0.11

0.27 0.13


Ratio

2.26

1.30

1.22

0.83

1.05

1.26

1.38

1.30

--


--


0.70

1.35

0.47

0.48


1.75

1.04

1.09

2.00



1.27





































Fig. 10. Chromatograms of amino acid constituents of leaves
at optimum and high temperature. (Third period of sampling.)





4















Add


Optimum Temperature


Aloninm


Isoleucine


Loucine


Glutomic Acid


High Temperature


Alonine


Valine


Leucln*


S.8


Serine
+Glutomine
+Asporagine


Effluent (m)


FIG. 10








84

at the optimum condition showed a significant decrease while

at the high temperature condition the total amino acids did

not change since the previous sampling period. The ratio of

total amino acids of high to optimum temperature was 21:13 =

1.6. The amounts of methionine sulfoxide and also total

amide content at the optimum condition showed a sharp de-

crease since the last sampling period. Total amide in the

plants grown at optimum temperature was one-sixth of those

at the high temperature (Table 9 and Figure 11). The un-

known A increased at high temperature, while it was absent

at the optimum temperature condition. There was a substantial

increase in homoserine content at both conditions. Lysine

was very low (trace) at high temperature while histidine was

very low at the optimum temperature condition.

5. Fifth period of sampling.-Samples were taken from
plants one week after their full flowering period (one week

after the previous sample). Plants under optimum temperature

had set small fruits while very few if any fruit could be
observed on the high temperature plants. There was little

change in total amino acid content of the plants under high
temperature conditions, while there was a considerable de-
crease at the optimum temperature condition. Total amino
acid content in the high temperature plants was twice that

under optimum conditions. There was a decrease of amide,

such that just a trace was left at the optimum condition












Amino Acid Content of


Amino acid

Methionine sulfoxide

Aspartic acid

Threonine

Serine

Asparagine

Glutamine

Homoserine

Glutamic acid

Proline

Unknown A

Glycine

Alanine

Valine

Isoleucine

Leucine

Tyrosine plus
phenylalanine

r-aminobutyric acid

Lysine

Histidine

Arginine


TABLE 9

Leaves Fourth Period of Sampling


0.27

0.63

0.20

Trace

0.38


0.44

1.14

Trace

0.33

0.16


).. '


pM per gram fresh weight
Temperature
Optimum High

0.40 0.91

1.05 1.96

0.41 0.34

1.20 1.43

0.29 2.05

0.10 0.50

2.63 4.87

2.53 3.27

Trace Trace

-- 0.32

0.14 0.11

1.85 2.42

0.57 0.57

0.13 0.28

0.12 0.22


--

0.78

1.31

1.00

2.15

1.83


1.62

2.80


0.42


I


Ratio

2.27

1.86

0.82

1.19

7.06

5.00

1.85

1.29


c.
I


,

































Fig. 11. Chromatograms of amino acid constituents of leaves
at optimum and high temperature. (Fourth period of sampling.)






* t ,'"3 -8


" .' ; ., '


Optimum Temperature


Glutomic Add


Alanine eIn


Leucine


Effluent (ml)


Efflunt (ml.)


FIG. 11


Serine
+6luoamln
+Asporoglne


r,







88

.while under the high temperature condition there was a large

amount of amide. Methionine sulfoxide was also decreased in

the optimum condition, while it was still high at high

temperature conditions (Table 10 and Figure 12). The only

amino acid which was increased at both temperatures was

homoserine. At the optimum temperature condition, the amount

of aspartic acid was very low while glutamic acid and alanine

were not decreased. At the high temperature there was only

a little decrease in aspartic acid. The unknown A was very

high at the high temperature (higher than in any other

sampling period), while at the optimum temperature it was

absent the same as in the previous sample. At high tempera-

ture, lysine, which was absent in the previous sample, was

again present while histidine became very low at both

temperature conditions.

III. Protein Estimation

The results of protein measurements in leaves of the

plants under high and optimum temperature conditions, for

five different periods of sampling, are given in Table 11

and in Figure 17.

The results are presented in terms of mg equivalent

of bovine serum albumin per gm of fresh weight. Protein

determinations were not from the same plant materials that

were used for amino acid studies but rather from the same


,11 1,









TABLE 10

Amino Acid Content of Leaves Fifth Period of Sampling


Amino acid

Methionine sulfoxide

Aspartic acid

Threonine

Serine

Asparagine

Glutamine

Homoserine

Glutamic acid

Proline

Unknown A

Glycine

Alanine

Valine

Isoleucine

Leucine

Tyrosine plus
phenylalanine

b-aminobutyric acid

Lysine

Histidine

Arginine


0.19

0.72

0.24

Trace

0.12


0.81

1.20

0.14

Trace

Trace


pM per gram fresh weight
Temperature
Optimum High

' 0.16 0.60

0.34 1.30

0.36 0.58

0.92 1.80

0.14 ? 1.14

Trace 0.34

3.45 2.97

1.63 2.73

Trace Present

0.65

0.43 0.24

1.55 2.92

0.23 0.52

0.12 0.23

0.06 0.07


Ratio

3.75

3.82

1.61

1.95

8.14



8.60

1.67


--


0.55

1.88

2.26

1.91

1.16


4.20

1.56

0.58
































Fig. 12. Chromatograms of amino acid constituents of leaves
at optimum and high temperature. (Fifth period of sampling.)









91
L8



t4monrine


Optimum Temperature
12-


1.0-

Serine
GluIomine Glutonic AeWi Alonine
A +Asparogine

.6-
Asportic Th,, Glycn Voine

Methionine
Sulfoxide \ Isoleucine Leucine



-- .2N No Citrote Buffer, pH 3.25 .-, .-2N No Citrote Buffer. H 4.25-
100 2io 140 10 W W 220 240 260 280 300 320 340 360 380400
Effluent (ml.)








> r,*


High Temperature


FIG. 12










variety of plants which were grown later under (as nearly

as possible) the same conditions. In the first sample,

which was taken seven days after the seeds were planted,

protein content was high in plants under both temperature

regimes but it was a bit higher in the optimum temperature.

High protein content at this sample period could result

from solublization and translocation of proteins from the

cotyledons to the young, yet not completely expanded leaves.

At the second sample which was taken two weeks after planting,

there was a sharp decrease in protein concentration of plants

grown at the two temperature conditions, when compared with

the first sample. Again, the protein concentration was

higher under the optimum than at the high temperature con-

dition.

The third sampling was made 21 days after planting at

high temperature and 23 days after planting at optimum con-

ditions in order to sample at'the same plastochron index

(P.I.). At this sampling period there was a sharp increase
in protein content of the plantsat the two conditions.

Once again the .total protein,of the plants at the optimum

temperature condition was higher than that of the plants at

the high temperature regime. The great increase in protein

content over the previous sampling may have resulted from:

increased photosynthetic activity; a decrease in the rate

of growth (under both temperature conditions); and finally,




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